CHIP下调导致CIB1泛素化受抑促进肺腺癌细胞EMT的机制研究

Lung adenocarcinoma is the most common pathological type of lung cancer. Epithelial mesenchymal transition (EMT) is closely related to invasion and metastasis of lung adenocarcinoma. Ubiquitination is widely involved in the development of tumors. Our previous studies have shown that CIB1 is highly expressed in lung adenocarcinoma and associated with its progression. CIB1 silence of lung adenocarcinoma cell inhibits TGFβ1 mediated E-cadherin downregulation and EMT. Immunoprecipitation and liquid chromatography tandem mass spectrometry shows that ubiquitin ligase CHIP interactes with CIB1. Previous experiment shows that CHIP expresses low in lung adenocarcinoma and interference of CHIP inhibits CIB1 ubiquitination and degradation and promotes E-cadherin downregulation. However it remains unclear that how does CHIP mediate CIB1 ubiquitination and degradation to regulate EMT in lung adenocarcinoma. Based on preliminary studies, we hypothesized that CIB1 is the target of ubiquitin ligase CHIP. CHIP regulates TGFβ1 induced EMT by mediating the stability of CIB1 via ubiquitination of CIB1 protein lysine residues. In this project, we propose to explore the potential mechanism of CHIP mediated CIB1 ubiquitination involved in the regulation of EMT in lung adenocarcinoma by using molecular biology, cell biology and animal experiment technology, and aim to find theoretical basis for new therapeutic targeted drugs for lung adenocarcinoma.

肺腺癌是肺癌最常见的病理类型，上皮间质转化（EMT）与肺腺癌侵袭及转移密切相关。泛素化广泛参与肿瘤的发生发展。我们前期发现CIB1在肺腺癌中高表达且与其进展相关，沉默CIB1可抑制TGFβ1介导E-cadherin的下调和EMT的发生。免疫共沉淀和液相色谱串联质谱分析显示，泛素连接酶CHIP与CIB1存在相互作用。预实验提示CHIP在肺腺癌中低表达，干扰CHIP可抑制CIB1泛素化与降解，促进E-cadherin的下调。但CHIP是如何介导CIB1泛素化与降解调控肺腺癌EMT发生的机制不清。据前期研究我们假设：CIB1是泛素连接酶CHIP的靶蛋白，CHIP通过对CIB1蛋白赖氨酸残基进行泛素化来调节其稳定性，从而调控TGFβ1介导EMT的发生。项目拟采用分子生物学、细胞生物学及动物实验技术进行研究，阐明CHIP介导CIB1泛素化调控肺腺癌EMT的机制，为研发肺腺癌新型靶向药物提供理论基础。

钙和整联蛋白结合1（CIB1）是调节细胞粘附，迁移和分化的钙调蛋白的同源物。它被认为是许多肿瘤细胞中的癌基因。但是，尚未研究其在肺腺癌（LAC）中的作用。本研究采用免疫组织化学方法检测LAC组织及邻近正常组织中CIB1的表达水平，并分析CIB1表达与患者临床病理特征的关系。在体外和体内测定了CIB1对LAC细胞的上皮-间质转化（EMT），迁移和转移的影响。使用电喷雾质谱（LS-MS）鉴定了与CIB1相互作用的蛋白质，并在以下测定中选择了CHIP。 Hsp70相互作用蛋白（CHIP）的羧基末端是泛素E3连接酶。我们表明，CHIP可以通过促进CIB1的多泛素化及其随后的蛋白酶体降解来降解CIB1。此外，CIB1的赖氨酸残基10和65是CIB1的泛素化位点。此外，CHIP介导的CIB1下调对于抑制LAC的转移和迁移至关重要。这些结果表明，CHIP介导的CIB1泛素化可以调节LAC中的上皮-间质和肿瘤转移。